Computer image analysis of variance between human chromosome replication sequences and G-bands.

A computer image analysis system was applied to the quantitative study of chromosomal early- and late-replication patterns from the leukocytes of several normal human donors, and these patterns were compared with the chromosomal G-banding patterns. The first and last few hours of replication were discriminated by selective bromodeoxyuridine vs. thymidine incorporation in DNA and a Hoechst-blacklight-Giemsa stain technique. Image analysis with Tufts Piquant system involved automatic determination of chromosome boundaries, centromeres and telomeres, linear chromatid axes, chromatid density measurements along each axis, and comparative length normalized density profiles for each chromatid and the chromosome. Consistent complementary early- and late-replication patterns were determined for autosomes 1-6 and the X chromosomes. Limited intracellular or interindividual variability occurred in the intensity of a few active replication peaks but not in their location. However, there were very distinct regions of noncorrespondence between the late-replication patterns and the G-band patterns, in contrast with previous observations, although many similarities were also evident. These differences are interpreted with reference to a general model of replication sequence control of cell differentiation.     

Temporal replication of an interspersed repeated sequence of mouse DNA

The temporal replication profile of an interspersed repeated DNA sequence (variously named MIF-1, Bam and L1Md) of mouse was determined by isotope analysis of a resolvable restriction fragment differentially labeled in pre- and post-synchrony cultures. While the temporal replication profile of the fragment was similar to that of total nuclear DNA, an average time lag of about 20 min was evident for this interspersed repeated family (called Bam in this paper). In addition, the sequence organisation of Bam homologues were examined for the separable early- and late-replication domains of the hamster genome. The data suggest that late-replicating domains of the rodent genome are slightly enriched in Bam homologous sequences. Furthermore, this repeated sequence family has different sequence organisations in the separable replication domains of hamster.     

DNA replication,3H-cRNA in situ hybridization and C-band patterns in the polycentric chromosomes ofLuzula purpurea Link

DNA late-replication,³H-cRNA in situ hybridization, and C-band distribution patterns were studied inLuzula purpurea Link chromosomes (2n=6). With each technique it was possible to identify homologous chromosomes. DNA late-replicating regions were present at the ends and in the middle of one chromosome pair (pair 1), on both ends of another chromosome pair with one end having more late-replicating regions than the other end (pair 2), and all along the length of the final pair (pair 3). The distribution of label following in situ hybridization of³H-cRNA complementary to Cot 1-reassociated DNA was similar to the DNA late-replication patterns. One chromosome pair had grains concentrated at the ends and in the middle of the chromosomes; another pair had grains at both ends with a greater grain concentration at one end; the final chromosome pair had grains distributed all along the length. C-band distribution patterns were also similar to the DNA late-replication and³H-cRNA in situ-hybridized ones. The results demonstrate that the constitutive heterochromatin ofL. purpurea polycentric chromosomes is similar to the constitutive heterochromatin of monocentric animal chromosomes in that it consists of highly repeated DNA sequences which are replicated late in the S stage of interphase.     

Patterns of late chromosomal DNA replication in unbalanced chinese hamster-mouse somatic cell hybrids

The chromosome late-replication patterns of five mouse × Chinese hamster somatic cell hybrids with reduced hamster complements were compared with those of the Chinese hamster parent cell, in order to determine whether the sequential order of chromosome replication is dependent on the presence of the whole chromosome set. In all hybrid clones the parental pattern could be recognized in a variable proportion of cells, although different chromosomes were missing in each clone. The results suggest that sequential order of replication is not the consequence of any sort of interaction between replication units (such as competition for limiting factors), and point to a considerable degree of autonomy in replication of individual chromosomes or chromosome parts.     

Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC signifi-cantly delayed the progression of the S phase and caused the phosphorylation of the Chk 1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern II was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-S-phase checkpoint blocks the DNA replication progression in the late stage of S phase.     

Reversal of DNA methylation with 5-azacytidine alters chromosome replication patterns in human lymphocyte and fibroblast cultures

Prior studies demonstrated that developmental or induced methylation of DNA can inactivate associated gene loci. Such DNA methylation can be reversed and specific genes reactivated by treatment with 5-azacytidine (5- azaC ). The present cytogenetic studies using replication banding methods show that 5- azaC treatment also results in an increase or decrease in replication staining at one or more band locations in human lymphocyte and fibroblast chromosomes. New replication band locations are not formed. These changes in replication staining, which reflect changes in timing of replication, are different between these two tissues. However, in both tissues, the delayed onset of replication in the heterocyclic, inactive X is shortened by 5- azaC . A correlation is thus suggested between the induced temporal change to earlier DNA replication, and induced hypomethylation and gene activation. The temporal effect on chromosome replication in 5- azaC -treated cells depends on the portion of the S-period studied. Toward the beginning of S, early-replication patterns are increased in both lymphocytes and fibroblasts. Toward the end of S, late-replication patterns are increased only in lymphocytes, suggesting a differential effect of 5- azaC in: (1) early-vs. late-S, and (2) lymphocytes vs. fibroblasts. Generally, 5- azaC has its greatest effect on the inactive chromosome regions that are typically late-replicating prior to 5- azaC treatment. These observed changes in replication band staining suggest that DNA methylation may modify regional groups of genes in concert.     

Transduction, Restriction and Recombination Patterns in Escherichia Coli

Chromosomal DNA from several Escherichia coli reference (ECOR) strains was transduced by bacteriophage P1 into E. coli strain K12 W3110 trpA33. Recombination patterns of the transductants were determined by restriction fragment length polymorphism over a 40-kb region centering on a single marker (trpA(+)) in the tryptophan operon. These experiments demonstrate that transduction between different strains of E. coli can result in recombinational replacements that are small in comparison to the entrant molecule (replacements average 8-14 kb, whereas P1 packages ~ 100 kb) often in a series of discrete segments. The transduction patterns generated resemble the natureal mosaic sequence patterns of the ECOR strains described in previous work. Extensive polymorphisms in the restriction-modification systems of the ECOR strains are a possible explanation for the sequence patterns in nature. To test this possibility, two transductants were back-transduced into strain K12 W3110 trpA33. The resulting patterns were strikingly different from the original transductions. The size of the replacements was greater, and no multiple replacements were observed, suggesting a role for restriction-modification systems in the transduction patterns and perhaps for the mosaic sequence patterns in nature.     

Heterogeneity of constitutive heterochromatin in somatic Syrian hamster chromosomes.

Syrian hamster constitutive heterochromatin was analyzed for C-band distribution and for BrU late-replication pattern. Characteristic for this species is relatively large amounts of sex-chromosome and autosomal heterochromatin. The distribution of constitutive heterochromatin was determined. The long term of the X chromosome, the whole Y, the short arms of 8 autosomal pairs, the long arm of the smallest metacentric pair, and the centromeric regions of 12 pairs stained intensely dark on C-band preparations. In contrast to the heterochromatin in the centromeric regions, the autosomal short-arm heterochromatin has an increased susceptibility to the denaturation process, as indicated by prolonged exposure to NaOH or Ba(OH)2. Such further exposure to denaturing agents results in an intense dark stain only on the sex-chromosome heterochromatin and centromeric regions of the autosomes. The BrdU late-replication pattern demonstrated that the late-replicating regions correspond to C-bands. Centromeric regions replicate late in the S phase; however, no centromeric region is among the latest replicating segments of the complement. Centromeric and noncentromeric heterochromatin are two distinct categories of constitutive heterochromatin.     

Analysis of loop boundaries using different local structure assignment methods

Loops connect regular secondary structures. In many instances, they are known to play important biological roles. Analysis and prediction of loop conformations depend directly on the definition of repetitive structures. Nonetheless, the secondary structure assignment methods (SSAMs) often lead to divergent assignments. In this study, we analyzed, both structure and sequence point of views, how the divergence between different SSAMs affect boundary definitions of loops connecting regular secondary structures. The analysis of SSAMs underlines that no clear consensus between the different SSAMs can be easily found. Because these latter greatly influence the loop boundary definitions, important variations are indeed observed, that is, capping positions are shifted between different SSAMs. On the other hand, our results show that the sequence information in these capping regions are more stable than expected, and, classical and equivalent sequence patterns were found for most of the SSAMs. This is, to our knowledge, the most exhaustive survey in this field as (i) various databank have been used leading to similar results without implication of protein redundancy and (ii) the first time various SSAMs have been used. This work hence gives new insights into the difficult question of assignment of repetitive structures and addresses the issue of loop boundaries definition. Although SSAMs give very different local structure assignments capping sequence patterns remain efficiently stable.     

Unusual chromatographic behavior of oligonucleotide sequence isomers on two different anion exchange HPLC columns

The retention behavior of the unmodified phosphodiester oligonucleotide sequence isomers was investigated on two different anion exchange columns: Biospher GMB 1000Q (based on DEAE-modified glycidyl methacrylate) and PolyWAX LP (based on silica with a crosslinked coating of linear polyethyleneimine). There was a notable difference in retention of oligonucleotides of the same composition but differing in the position of a single base. The most pronounced difference was observed between the oligonucleotides with the variable base in the end and in the center of the sequence. The use of either acetonitrile or 2-propanol as a mobile phase organic modifier did not markedly affect the retention time patterns. Prediction of the retention times of oligonucleotides must take into account the base position as well as identity. This is the first report of such a "same composition different sequence" effect, described for the short peptides, for synthetic oligonucleotides.     

Screening strategies for a highly polymorphic gene: DHPLC analysis of the Fanconi anemia group A gene

Introduction: Patients with Fanconi anemia (Fanc) are at risk of developing leukemia. Mutations of the group A gene (FancA) are most common. A multitude of polymorphisms and mutations within the 43 exons of the gene are described. To examine the role of heterozygosity as a risk factor for malignancies, a partially automatized screening method to identify aberrations was needed. We report on our experience with DHPLC (WAVE (Transgenomic)). Methods: PCR amplification of all 43 exons from one individual was performed on one microtiter plate on a gradient thermocycler. DHPLC analysis conditions were established via melting curves, prediction software, and test runs with aberrant samples. PCR products were analyzed twice: native, and after adding a WT-PCR product. Retention patterns were compared with previously identified polymorphic PCR products or mutants. Results and discussion: We have defined the mutation screening conditions for all 43 exons of FancA using DHPLC. So far, 40 different sequence variations have been detected in more than 100 individuals. The native analysis identifies heterozygous individuals, and the second run detects homozygous aberrations. Retention patterns are specific for the underlying sequence aberration, thus reducing sequencing demand and costs. DHPLC is a valuable tool for reproducible recognition of known sequence aberrations and screening for unknown mutations in the highly polymorphic FancA gene.     

Disentangling confusions in inflorescence morphology: Patterns and diversity of reproductive shoot ramification in angiosperms

Terminology of inflorescence diversity has often been used in a confusing way in the literature, partly because it was based on uncritical and outdated definitions. In particular, the terms cyme, thyrse, and panicle have been misused. Although a more critical classification worked out by several authors is available, it is unfortunately not in general use because most of the relevant publications are written in German. In addition, some terms have not been used in the same way by morphologists and developmental geneticists. The present review attempts to remedy the situation with a simple outline of a classification based on: (i) different branching patterns; (ii) differential elongation of axes of different orders; and (iii) repetition of basic ramification patterns in different ways. Racemose and cymose branching are two extreme patterns; the former with limitation of axial orders to two, the second with limitation of lateral axes of each order to two. In a branching system, a sequence of racemose → cymose and, within the cyme, of dichasial → monochasial is common, but the reverse sequence generally does not occur. Systematic and evolutionary aspects of inflorescences are briefly discussed. Branching patterns are often stable in larger clades.Infiorescences of mutants studied in developmental genetic studies are mainly altered in flower or branch numbers or relative branch length, but not in branching patterns. This is also a contribution towards the goal of a unified terminology for the different fields of biology dealing with inflorescences.     

Disentangling confusions in inflorescence morphology:Patterns and diversity of reproductive shoot ramification in angiosperms

Terminology of inflorescence diversity has often been used in a confusing way in the literature,partly because it was based on uncritical and outdated definitions.In particular,the terms cyme,thyrse,and panicle have been misused.Although a more critical classification worked out by several authors is available,it is unfortunately not in general use because most of the relevant publications are written in German.In addition,some terms have not been used in the same way by morphologists and developmental geneticists.The present review attempts to remedy the situation with a simple outline of a classification based on:(i)different branching patterns;(ii)differential elongation of axes of different orders;and(iii)repetition of basic ramification patterns in different ways.Racemose and cymose branching are two extreme patterns;the former with limitation of axial orders to two,the second with limitation of lateral axes of each order to two.In a branching system,a sequence of racemose→cymose and,within the cyme,of dichasial→monochasial is common,but the reverse sequence generally does not occur.Systematic and evolutionary aspects of inflorescences are briefly discussed.Branching patterns are often stable in larger clades.Inflorescences of mutants studied in developmental genetic studies are mainly altered in flower or branch numbers or relative branch length,but not in branching patterns.This is also a contribution towards the goal of a unified terminology for the different fields of biology dealing with inflorescences.     

Repetitive sequences in Eurasian lynx (Lynx lynx L.) mitochondrial DNA control region

Mitochondrial DNA (mtDNA) control region (CR) of numerous species is known to include up to five different repetitive sequences (RS1-RS5) that are found at various locations, involving motifs of different length and extensive length heteroplasmy. Two repetitive sequences (RS2 and RS3) on opposite sides of mtDNA central conserved region have been described in domestic cat (Felis catus) and some other felid species. However, the presence of repetitive sequence RS3 has not been detected in Eurasian lynx (Lynx lynx) yet. We analyzed mtDNA CR of 35 Eurasian lynx (L. lynx L.) samples to characterize repetitive sequences and to compare them with those found in other felid species. We confirmed the presence of 80 base pairs (bp) repetitive sequence (RS2) at the 5' end of the Eurasian lynx mtDNA CR L strand and for the first time we described RS3 repetitive sequence at its 3' end, consisting of an array of tandem repeats five to ten bp long. We found that felid species share similar RS3 repetitive pattern and fundamental repeat motif TACAC.     

The structure of a subterminal repeated sequence present on many human chromosomes.

All telomeres which have been studied consist of an array of simple G/C rich repeats. Human telomeres were shown to share sequence similarity with those of lower eukaryotes by cross-hybridization and human telomeric sequences have been cloned by complementation of telomere function in yeast. Analysis of human telomeric sequences cloned in this way is described here. The terminal part of the cloned human telomeric DNA consists of an array of simple repeats, principally of the sequence TTAGGG and derivatives. The very terminal part consists of yeast-type telomeric repeats which suggests that the human telomeric sequences have acted as a primer for the addition of additional telomeric repeats in the yeast. Subterminal sequences are shared between a number of clones and in situ data shows that these subterminal sequences are present at several different chromosomal ends. Related sequences are present at internal as well as telomeric positions. Differences in the hybridization patterns of subterminal sequences in somatic compared to germ-line tissues are described which indicate differential modification of these sequences during development.     

Predatory bug <Emphasis Type="Italic">Picromerus bidens</Emphasis> communicates at different frequency levels

The Asopinae (Heteroptera: Pentatomidae) are a subfamily of stinkbugs with predaceous feeding habits and poorly understood communication systems. In this study we recorded vibratory signals emitted by Picromerus bidens L. on a non-resonant substrate and investigated their frequency characteristics. Males and females produced signals by vibration of the abdomen and tremulation. The female and male songs produced by abdominal vibrations showed gender-specific time structure. There were no differences in the temporal patterns of male or female tremulatory signals. The signals produced by abdominal vibrations were emitted below 600 Hz whereas tremulatory signals had frequency ranges extending up to 4 kHz. Spectra of male vibratory signals produced by abdominal vibrations contained different peaks, each of which may be dominant within the same song sequence. Males alternated with each other during production of rivalry signals, using different dominant frequency levels. We show that the vibratory song repertoire of P. bidens is broader than those of other predatory stinkbugs that have been investigated. The emission of vibrational signals with different dominant frequencies but the same production mechanism has not yet been described in heteropteran insects, and may facilitate location of individual sources of vibration within a group.     

Identification of function-associated loop motifs and application to protein function prediction

MOTIVATION: The detection of function-related local 3D-motifs in protein structures can provide insights towards protein function in absence of sequence or fold similarity. Protein loops are known to play important roles in protein function and several loop classifications have been described, but the automated identification of putative functional 3D-motifs in such classifications has not yet been addressed. This identification can be used on sequence annotations. RESULTS: We evaluated three different scoring methods for their ability to identify known motifs from the PROSITE database in ArchDB. More than 500 new putative function-related motifs not reported in PROSITE were identified. Sequence patterns derived from these motifs were especially useful at predicting precise annotations. The number of reliable sequence annotations could be increased up to 100% with respect to standard BLAST. CONTACT: boliva@imim.es SUPPLEMENTARY INFORMATION: Supplementary Data are available at Bioinformatics online.     

Komplementäre DNA-Replikationsmuster bei Drosophila melanogaster

In D. melanogaster salivary gland chromosomes continuous and discontinuous labeling patterns have been described. Arcos-Terán (1972) has devided those of the X-chromosome in into a 6 class sequence beginning with continuous labeling and ending with labeling restricted to a few heavy bands. Labeling patterns reverse to the described ones have not been reported. — In the present communication new types of discontinuous labeling patterns are described for the X of melanogaster females. One type shows unlabeling of the majority of heavy bands and of the chromocenter whereas interbands and faint bands are labeled. In other types the number of unlabeled heavy bands is decreased and the chromosome is nearly totally labeled. These new labeling patterns are the reverse of Arcos-Terán's patterns II, IV, and V. It must therefore be assumed that the continuous pattern of labeling corresponds to the (early) middle of the replication cycle and that replication in melanogaster females starts with the labeling patterns described here.     

TIT for TAT: the properties of inosine and adenosine in TATA box DNA

The sequence dependent conformation, flexibility and hydration properties of DNA molecules constitute selectivity determinants in the formation of protein-DNA complexes. TATA boxes in which AT basepairs (bp) have been substituted by IC bp (TITI box) allow for probing these selectivity determinants for the complexation with the TATA box-binding protein (TBP) with different sequences but identical chemical surfaces. The reference promoter Adenovirus 2 Major Late Promoter (mlp) is formed by the apposition of two sequences with very different dynamic properties: an alternating TATA sequence and an A-tract. For a comparative study, we carried out molecular dynamics simulations of two DNA oligomers, one containing the mlp sequence (2 ns), and the other an analog where AT basepairs were substituted by IC basepairs (1 ns). The simulations, carried out with explicit solvent and counterinons, yield straight purine tracts, the A-tract being stiffer than the I-tract, an alternating structure for the YRYR tracts, and hydration patterns that differ between the purine tracts and the alternating sequence tracts. A detailed analysis of the proposed interactions responsible for the stiffness of the purine tracts indicates that the stacking between the bases bears the strongest correlation to stiffness. The hydration properties of the minor groove in the two oligomers are distinctly different. Such differences are likely to be responsible for the stronger binding of TBP to mlp over the inosine-substituted variant. The calculations were made possible by the development, described here, of a new set of forcefield parameters for inosine that complement the published CHARMM all-hydrogen nucleic acid parametrization.